Actobindin, an 88 amino acid protein from Acanthamoeba castellanii that is capable of binding two actin monomers, is a potent inhibitor of the nucleation phase of actin polymerization at concentrations substantially less than the actin concentration. Recent data have shown that actobindin binds more tightly to covalently cross-linked "upper" dimer than predicted if the dimer were simply a bivalent ligand with two sites of the same affinity for actobindin as actin monomer. Actobindin binds to cross- linked "lower" dimer with lower affinity. Cross-linking actin during normal polymerization produces both the lower and upper dimer but when actobindin is present only the cross-linked lower dimer is formed. The amount of cross-linked lower dimer formed in the presence of actobindin is proportional to the square of the actin concentration. These, and other, data suggest that a native analogue of the cross-linked upper dimer may be the polymerization intermediate with which actobindin interacts and that the interaction either causes this dimer to dissociate or to convert to the native analogue of the cross-linked lower dimer. In either case, the presence of actobindin would block the formation of the upper dimer analogue and increase the formation of the lower dimer analogue that would not be on the polymerization pathway.